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J Reprod Dev
2019 Oct 23;655:407-412. doi: 10.1262/jrd.2019-040.
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Expression and function of exportin 6 in full-grown and growing porcine oocytes.
Onuma A, Fujioka YA, Fujii W, Sugiura K, Naito K.
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Exportin 6, which functions specifically in the nuclear export of actin family proteins, has been reported to be absent in immature Xenopus oocytes, which have a huge nucleus containing a large amount of actin. In mammalian oocytes, however, the presence and the function of exportin 6 remain uninvestigated. In this study, we assessed the expression and effects of exportin 6 on meiotic resumption in porcine oocytes after cloning porcine exportin 6 cDNA and carrying out overexpression and expression inhibition by mRNA and antisense RNA injection, respectively. We found for the first time that exportin 6 was expressed in mammalian full-grown germinal-vesicle-stage oocytes and was involved in the nuclear export of actin. In contrast, exportin 6 was absent from the growing oocytes, which are meiotically incompetent and maintain the germinal-vesicle structure in the long term; the regulatory mechanism appeared to be active degradation. We examined the effects of exportin 6 on meiotic resumption of porcine oocytes and noted that its expression did not affect the onset time but increased the rate of germinal vesicle breakdown at 24 h via regulation of the nuclear actin level, which directly influences the physical strength of the germinal-vesicle membrane. Our results suggest that exportin 6 affects the nuclear transport of actin and meiotic resumption in mammalian oocytes.
Fig. 1. Expression and turnover of endogenous XPO6 in porcine growing (GO) or full-grown (FGO) oocytes. (A) The XPO6 protein expression was examined via immunoblotting. Fifteen oocytes were
used without culture (GV) and 15 were used after 48 h of maturation culture (MII). A representative result is presented in the upper panel. Beta-actin (ACTB) was used as a loading
control. The XPO6 expression levels are presented as the ratio to ACTB in the lower panel (mean ± SD, * P < 0.05 compared with GV of FGOs). (B) The mRNA expression of XPO6 was
analyzed via RT-PCR. RPL19 was used as a loading control. (C) The turnover of endogenous XPO6 in FGOs was analyzed via immunoblotting. Meiotic resumption and protein synthesis were
inhibited by roscovitine (Ros) and cycloheximide (CHX), respectively. Fifteen oocytes were loaded in each lane.
Fig. 2. Overexpression and nuclear-export function of XPO6 in porcine growing (GO) and full-grown (FGO) oocytes. (A) Oocytes with or without XPO6 or XPO1
mRNA injection were cultured for 24 h and the expression of XPO6 (left panel) or XPO1 (right panel) was examined via immunoblotting. Fifteen oocytes were used for each lane. Beta-actin
(ACTB) was used as a loading control. (B) Calculation method for the nuclear localization index of ACTB. A rectangle (broken line) was drawn on the oocyte diameter including the
nucleus with the fixed lengthwise using ImageJ software as presented in the upper part of the image. The obtained plot represented in the lower part indicates the average fluorescence
intensity of the lengthwise dimension of the rectangle. The nuclear and cytoplasmic averages of the fluorescence intensity were calculated as the quotient of their area (gray, nucleus
(N); stripe, cytoplasm (C)) divided by their width. The part of the nucleolus was excluded for calculating the nuclear average. The ratio of fluorescence intensity in the nucleus to
that in the cytoplasm was obtained from each oocyte and used as the nuclear localization index. (C) Representative localization of ACTB with and without XPO6 overexpression was
examined via immunocytochemistry. DNA was stained by 4’,6-diamidino-2-phenylindole. Scale bar, 50 µm. (D) The nuclear localization index of ACTB was calculated as the ratio of
fluorescence intensity in the nucleus to that in the cytoplasm as described in (B). More than eight oocytes were analyzed in each group (mean ± SEM, * P < 0.05).
Fig. 3. Effect of XPO6 on meiotic resumption of porcine full-grown oocytes. (A) The GVBD rate of the XPO6 mRNA-injected oocytes was examined at the indicated culture periods
(* P < 0.05). The oocytes into which only EGFP mRNA was injected were used as a control. The number of oocytes examined in every experimental group was indicated in
each bar. Results were shown as mean ± SEM. (B) The XPO6 expression in oocytes with or without XPO6 antisense RNA (asXPO6)-injection was analyzed via
immunoblotting. The oocytes were cultured for 48 h with roscovitine (Ros) for GV-stage maintenance. Fifteen oocytes were used for each lane. (C) The asXPO6-injected
oocytes were precultured for 48 h with Ros to decrease endogenous XPO6. After the preculture, the oocytes were cultured without Ros and the GVBD rate was examined at the indicated
culture periods (* P < 0.05). The oocytes into which only EGFP mRNA was injected were used as the control. The number of oocytes examined in every experimental
group was indicated in each bar. Results are presented as mean ± SEM.
Baarlink,
A transient pool of nuclear F-actin at mitotic exit controls chromatin organization.
2017, Pubmed
Baarlink,
A transient pool of nuclear F-actin at mitotic exit controls chromatin organization.
2017,
Pubmed Bohnsack,
A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes.
2006,
Pubmed
,
Xenbase Caridi,
Nuclear F-actin and myosins drive relocalization of heterochromatic breaks.
2018,
Pubmed Clark,
Diffusible and bound actin nuclei of Xenopus laevis oocytes.
1977,
Pubmed
,
Xenbase Dessev,
A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.
1991,
Pubmed Dopie,
Active maintenance of nuclear actin by importin 9 supports transcription.
2012,
Pubmed Falahzadeh,
The potential roles of actin in the nucleus.
2015,
Pubmed Fornerod,
CRM1 is an export receptor for leucine-rich nuclear export signals.
1997,
Pubmed
,
Xenbase Fujioka,
Analyses of EMI functions on meiotic maturation of porcine oocytes.
2016,
Pubmed
,
Xenbase Fung,
Structural determinants of nuclear export signal orientation in binding to exportin CRM1.
2015,
Pubmed Görlich,
Transport between the cell nucleus and the cytoplasm.
1999,
Pubmed Hirao,
Association between p34cdc2 levels and meiotic arrest in pig oocytes during early growth.
1995,
Pubmed Kimura,
Biological significance of the importin-β family-dependent nucleocytoplasmic transport pathways.
2014,
Pubmed Kutay,
Leucine-rich nuclear-export signals: born to be weak.
2005,
Pubmed Lüscher,
A role for the p34cdc2 kinase and phosphatases in the regulation of phosphorylation and disassembly of lamin B2 during the cell cycle.
1991,
Pubmed Motlik,
Meiotic competence in vitro of pig oocytes isolated from early antral follicles.
1984,
Pubmed Mühlhäusser,
Multiple pathways contribute to nuclear import of core histones.
2001,
Pubmed Naito,
Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro.
1988,
Pubmed Nishimura,
Analyses of the involvement of PKA regulation mechanism in meiotic incompetence of porcine growing oocytes.
2012,
Pubmed Onuma,
Effects of exportin 1 on nuclear transport and meiotic resumption in porcine full-grown and growing oocytes.
2018,
Pubmed Pemberton,
Mechanisms of receptor-mediated nuclear import and nuclear export.
2005,
Pubmed Schrank,
Nuclear ARP2/3 drives DNA break clustering for homology-directed repair.
2018,
Pubmed
,
Xenbase Stade,
Exportin 1 (Crm1p) is an essential nuclear export factor.
1997,
Pubmed Stüven,
Exportin 6: a novel nuclear export receptor that is specific for profilin.actin complexes.
2003,
Pubmed Wang,
Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development.
2000,
Pubmed
